Nebulin is an integral protein of skeletal muscle thin laments and probably acts as a ruler for the thin lament length. Cardiac muscles of higher vertebrates have been shown earlier to lack nebulin. Instead in human and chicken cardiac muscle the much smaller protein nebulette replaces nebulin. Since nebulette is confined to the Z-disc region of the sarcomere and does not span the whole thin lament length, it must have functions significantly different from those assumed for nebulin. We have investigated nebulin in skeletal and cardiac muscles of the agnathans (lamprey, hagfish), elasmobranchs (shark), chondrosts (sturgeon) and teleosts (trout, eel) by SDS-PAGE and immunodetection methods. Among these, lamprey and hagfish cardiac muscles are unique in that both contain full-length nebulin identical in molecular mass to the nebulin of the respective body muscle. Using immuno fluorescence microscopy, lamprey cardiac nebulin was localised in the I-band of the sarcomere, the same as for nebulin in skeletal muscle. In contrast to this, all gnathostome species investigated lacked nebulin in cardiac muscles, while it was present in the respective skeletal muscles. This clearly shows that nebulin is not exclusively present in skeletal muscles of chordates. The findings also demonstrate a rare case of dramatic size reduction of a protein during evolution.